Diagnosis of thalassemia in ancient bones: 

 Problems and prospects in pathology 



Antonio Ascenzi, A. Bellelli, M. Brunori, G. Citro, R. Ippoliti, 

 E. Lendaro, and R. Zito 



Porotic hyperostosis" is a generic term applied to a type of 

 bone lesion characterized by a symmetrically distributed in- 

 crease in the volume of the skeleton, associated with a reduc- 

 tion of the bone texture. Such a type of lesion was recognized 

 for the first time by Cooley and Lee (1925) as a feature 

 peculiar to thalassemia. Subsequent investigations (Moseley 

 1963; Ascenzi 1976.1979) deeply modified the original 

 view, so that nowadays it is quite obvious that porotic hyper- 

 ostosis can be induced by any disease that leads to an increase 

 in bone marrow volume, causing the skeleton to adapt its 

 capacity to contain the excess of hemopoietic marrow. The 

 most profound changes of porotic hyperostosis are seen in 

 children, and they diminish as the individual approaches 

 adult life (Caffey 1951). This agrees with Neumann's law in 

 which half of the bone marrow is adipose in the adult, while 

 the entire long bone marrow is hemopoietic in the child. 

 Because of this, the adult can double the volume of hemo- 

 poietic marrow without any change to the skeleton; on the 

 other hand, even a limited hyperplasia of the hemopoietic 

 marrow induces in the subadult a volumetric increase in the 

 bone marrow. A list of conditions inducing porotic hyper- 

 ostosis has been compiled by Moseley (1965). and the sub- 

 ject has been reviewed by Ascenzi (1976,1979). In theory, 

 any condition that abnormally increases the rate of blood cell 

 turnover can produce porotic hyperostosis. Disea.ses known 

 to do this include: congenital hemolytic anemias (thalasse- 

 mia, sickle cell disease, hereditary spherocytosis, hereditary 

 elliptocytosis. hereditary nonspherocytic hemolytic anemia), 

 iron deficiency anemia, cyanotic congenital heart disease, 

 polycythemia vera in childhood. 



From what is reported above it may be inferred that, in 

 paleopathologic terms, porotic hyperostosis is the only suita- 

 ble evidence for the existence of medullary hyperplasia, ei- 

 ther primary or secondary, when the skeleton is the only 

 tissue which time has preserved. However, skeletal remains 

 showing porotic hyperostosis have been unable so far to pro- 

 vide unequivocal information on the specific disca.se syn- 

 drome which led to bone lesions. 



Zagreb PaUopathotogy Symp. 1988 



Starting with these premises, we attempted to remove am- 

 biguities for the diagnosis of thalassemia in skeletal remains 

 by examining the possibility that hemoglobin arising from 

 postmortem lysis of the erythrocytes may remain adsorbed to 

 the bone and be specifically detected. In a previous paper 

 (Ascenzi et al. 1985) we provided evidence that hemoglobin 

 is indeed measurable in skeletal remains dating back to En- 

 eolytic age using an immunochemical technique. This dis- 

 covery encouraged further investigations, and in this paper 

 we present additional progress toward an unequivocal diag- 

 nosis of thalassemia in skeletal remains with porotic hyper- 

 ostosis. 



Materials and methods 



Samples (lumbar vertebrae, skulls and other bones) were 

 obtained from the Verano cemetery of Rome (samples buried 

 for 15 to 30 years), from the S. Senatore Catacumbae in 

 Albano Laziale, Rome (samples dating back from the first to 

 thirdcentury of thisera), from the Necropolis of Porto (Isola 

 Sacra, Rome, second century B.C.) and of Castiglione 

 (Rome, 1000 B.C.). 



Bones were frozen in liquid nitrogen and crushed into 

 powder with a hydraulic press. The powder was extracted 

 with 6M urea for 4 hours at room temperature and neutral pH . 

 The extract was filtered and urea was removed by means of 

 gel permeation chromatography on Sephadex G25 (Phar- 

 macia, Sweden). 



Antiserum against human apohemoglobin (globin) was 

 raised in adult male rabbits with three subcutaneous inocula- 

 tions of 0.8 mg of the antigen in complete Freund adjuvant. 

 Hemoglobin constituent alpha and beta chains were prepared 

 as by Bucci and Fronticelli (1965) and antisera against the 

 two purified chains were obtained following the same pro- 

 cedure. The content of hemoglobin remnants in bone extracts 

 was determined with the immunoblot technique employing a 

 Bio Dot apparatus (BioRad, U.S.A.); solutions of apo- 

 hemoglobin, alpha and beta chains were employed as stan- 



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